Rapid dual direct fluorescent antibody assay for the identification of Bacillus antrhacis
Inventors
Abshire, Teresa • Ribot, Wilson J
Assignees
United States Department of the Army
Publication Number
US-9995746-B2
Publication Date
2018-06-12
Expiration Date
2034-04-02
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Abstract
In this application is described a method for rapidly and accurately identifying B. anthracis in a sample by simultaneously detecting the presence of cell wall antigen and capsule antigen in the same sample culture grown under capsule inducing conditions. Other uses and advantages of the method of the invention are described herein.
Core Innovation
The invention discloses a novel and rapid detection method for simultaneously identifying two main indicators of Bacillus anthracis: the cell wall polysaccharide Gal-NAG-PS and the capsule poly-D-glutamic acid. The method uses a Fab fragment of an anti-Gal-NAG-PS antibody, which, due to its small size, can permeate the B. anthracis capsule and bind to the cell wall antigen even under capsule-inducing conditions, unlike full-size IgG or IgM antibodies.
This method allows the simultaneous detection of capsule and cell wall antigens in a single culture under capsule-inducing conditions, eliminating the traditional need for multiple subcultures and parallel immunoassays for each antigen. The sample is incubated under capsule-inducing conditions, then contacted with two differently labeled antibodies—one Fab fragment specific for cell wall and the other specific for capsule—allowing detection by observing the specific signals for each antigen.
The background problem addressed by the invention is the inefficiency of current methods, which require two separate culture conditions and vials due to the inhibitory effect of the capsule on cell wall antigen detection by whole antibodies. This process is time-consuming, expensive, and increases the likelihood of misinterpretation. The new method streamlines the workflow, improves accuracy, reduces error, and enables more rapid identification of Bacillus anthracis in various sample types.
Claims Coverage
The patent contains two independent claims: one directed to a method for detecting Bacillus anthracis in a sample after culturing under capsule-inducing conditions, and another directed to a method for detecting Bacillus anthracis in uncultured vegetative form samples.
Simultaneous detection of capsule and cell wall antigens in a single sample under capsule-inducing conditions
The method comprises: 1. Culturing a single sample for 2-3 hours under conditions suitable to induce Bacillus anthracis capsule formation, resulting in a single cultured sample. 2. Directly contacting this single cultured sample in a single container with two antibodies in tandem, each detectably labeled with a different identifiable signal moiety, such that both antibodies are present in the same culture sample together. - One antibody specifically binds to the capsule antigen. - The second is a Fab antibody that specifically binds a cell wall antigen and has a radius sufficiently small to permeate the Bacillus anthracis capsule, enabling it to bind the cell wall antigen even in the presence of capsule-inducing conditions. 3. Detecting the presence of the detectable signals from both antibodies, with the simultaneous presence of both signals indicating the presence of Bacillus anthracis in the sample.
Detection of Bacillus anthracis in uncultured, intact vegetative cell samples using tandem antibodies
The method comprises: 1. Contacting a single, uncultured sample (with cells in intact vegetative form and less than 24 hours old) with two antibodies in tandem in a single container, each detectably labeled with a different identifiable signal moiety. - One antibody specifically binds to a capsule antigen. - The Fab antibody specifically binds to a cell wall antigen and has a sufficiently small radius to permeate the Bacillus anthracis capsule, binding to the cell wall antigen even in capsule-inducing conditions. 2. Detecting the presence of signals from both antibodies, with the simultaneous presence indicating the presence of Bacillus anthracis in the single sample.
The inventive features focus on a dual-antibody tandem assay that allows for simultaneous detection of both capsule and cell wall antigens in either cultured or direct vegetative form samples, using antibody fragments tailored for capsule penetration and differential labeling for rapid, accurate identification of Bacillus anthracis.
Stated Advantages
Eliminates the need for separate culture conditions for each antigen, enabling detection of both B. anthracis cell wall and capsule antigens in a single vial and under a single incubation.
Increases rapidity and accuracy of B. anthracis identification by enabling simultaneous detection and reducing time and cost.
Enables accurate detection of atypical isolates or aberrant Bacillus species that produce either capsule or anthracis-like cell wall polysaccharide.
Allows immediate analysis and early identification of Bacillus anthracis in incoming cultures or blood samples without further culturing.
Minimizes misinterpretation of results by staining the same bacillus with antibodies conjugated with different fluorescent labels.
Documented Applications
Detection and identification of Bacillus anthracis in biological, environmental, forensic, or food samples using the tandem DFA assay.
Use in test kits for detection of B. anthracis, comprising the specific antibodies labeled with detectable reporter molecules.
Targeting therapeutic compounds to Bacillus anthracis by complexing anti-Gal-NAG-PS and anti-CAP antibodies to therapeutic or bactericidal compounds for delivery to the cell wall or capsule.
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